Carcinogenesis

Question 1

DNA damaging factors: exogenous vs endogenous

Answer 1

1. Exogenous (extracellular)
   - UV light
   - X-rays
   - Natural isotopes
   - Chemicals (natural / man-made)
2. Endogenous (intracellular)
   - Oxygen
   - Water
   - Reactive metabolism

Question 2

How does UV light cause DNA damage

Answer 2

• Formation of cyclobutane pyrimidine dimers (thymine and CC dimers which interfere with base pairing during DNA replication)
• 6-4 photoproducts = covalent bond between the carbon at the 6 position of one ring and carbon at the 4 position of the ring on the next. This is frequent but more mutagenic than cyclobutane pyrimidine dimers
• UV damage causes DNA distortion and TT dimer is a signature

Question 3

Alkylating agents and DNA damage

Answer 3

Some are used to treat cancer because the idea is that cells that are replicating faster are more sensitive to these agents

• Alkylation of bases or multiple bases will break the helix again

Question 4

Endogenous DNA damage - aflatoxin

Answer 4

Exposure through eating contaminated food

Increased risk of liver cancer

Cytochrome p450 adds a highly reactive epoxide group, if not immediately disarmed with glutathione, it can attack DNA, leading to hydrolysis or breaks in DNA

Question 5

Different exogenous mutations of p53

Answer 5

• UV light (CC to TT (pyrimidine dimers))
• Aflatoxin B1 in diet (liver cancer)
• Benzoapyrene in tobacco smoke (lung cancer)

Question 6

How does endogenous mutation of p53 occur

Answer 6

Methylation of C at CpG (in gene promoter regions)

• Most p53 mutations are located at CpG sites

Question 7

How is alkylation damage repaired?

Answer 7

• Direct reversal
• Covalent transfer of the alkyl group from the modified base to a cysteine residue on the DNA repair enzyme
• Suicide enzyme such as MGMT which removes the alkylation, leading to the enzyme being turned over AKA dying

Question 8

Explain base excision repair (BER)

Answer 8

• Short patch repair (single nucleotides) or long patch repair (2-10 nucleotides)
• DNA glycosylases remove the base, producing an AP site (apurinic/apyrimidinic site = empty gap) which is then filled using AP endonucleases (these use the complementary strand to repair the gap)

Question 9

Explain nucleotide excision repair (NER)

Answer 9

Many forms of DNA are not recognised by DNA glycosylases and thus cannot be repaired by BER

• NER removes an oligonucleotide fragment that contains the damaged base or bases instead of a single base as in BER. This is more relevant to bigger lesions such as pyrimidine dimers or 6-4 photoproducts
• NER is mediated by the XPC complex. This is a DNA damage sensor complex which will recognise more important DNA damage/lesions. XPA binds to pyrimidine dimer and acts as a scaffold to recruit other proteins which will initiate repair!

Question 10

How does the XPC complex cause NER

Answer 10

1. XPA binds to pyrimidine dimer and acts as scaffold to recruit other proteins to the repair complex
2. XPB and XPD helicases separate DNA strands around the damaged area. RPA keeps strands apart
3. XPG and XPF endonucleases cut the DNA on either side of the damage
4. The cut fragment is removes, the gap is filled in by DNA polymerases and then sealed by DNA ligase

Question 11

Explain DSB repair

Answer 11

DSBs can arise spontaneously and through exposure to ionising radiation
- Cannot used complementary DNA strand to help restore the break

Repair via: non-homologous end joining and homologous recombination

Question 12

What is Xeroderma pigmentosum

Answer 12

Genetic disorder in which there is a defect in nucleotide excision repair of UV damage

• Causes extreme sensitivity to sunlight by approx 2y/o
• > 100x higher risk of skin cancer by approx 8y/o

Question 13

Explain the 1st and 2nd hit (mutation) of TSGs

Answer 13

• The TSG can gain a mutation which affects their function (inherited or sporadic) = 1st hit
• Eventually the whole gene will become silenced either through acquisition of a second hit (point mutation or a loss of heterozygosity) or hypermethylation (methylation of promoter so gene cannot be transcribed)

Question 14

Hereditary forms of colorectal cancer

Answer 14

1. FAP: Familial Adenomatous Polyposis
   - Polyposis
   - Autosomal dominant inheritance
   - APC gene - negative regulator of beta catenin (beta catenin pathway leads to cell growth)
   - More spontaneous
2. HNPCC: Hereditary Non Polyposis Colon Cancer
   - Lynch syndrome
   - No polyposis
   - More synchronous

Question 15

Explain the mechanism of HNPCC

Answer 15

• Direct mismatch repair deficiency by mutation of the two proteins MSH2 and MLH1
• So we get DNA replication errors, meaning that when you inherit DNA from one cell to another, there may be a mistake in the DNA such as ss breaks
• These two proteins are there to correct any mistakes regarding base mismatch or replication errors, so defects lead to mutations accumulating whenever a cell replicates, an accumulation of mutations leads to a ‘mutator phenotype’

Question 16

What is the mutator phenotype hypothesis

Answer 16

That at some point there is an event in a cancer cell, at a pre-cancerous stage, that it becomes susceptible to accumulating mutations. That’s why the rate of mutation of a cancerous cell is high, but in a normal cell it is extremely low